sensors

Article
Development of a Low-Cost UV-Vis
Spectrophotometer and Its Application for the
Detection of Mercuric Ions Assisted by Chemosensors
David González-Morales 1 , Asmilly Valencia 2 , Astrid Díaz-Nuñez 3 , Marcial Fuentes-Estrada 4 ,
Oswaldo López-Santos 1, * and Olimpo García-Beltrán 4, *
1 Facultad de Ingeniería, Universidad de Ibagué, Carrera 22 Calle 67, Ibagué 730002,
Colombia; david.gonzalez@unibague.edu.co
2 Facultad de Ingeniería Forestal, Universidad del Tolima, Altos de Santa Helena, Ibagué 730001,
Colombia; ayvalencial@ut.edu.co
3 Universidad Nacional de Colombia, Sede Medellín, Escuela de Química, Carrera 65, No. 59A-110,
Medellín 050034, Colombia; asdiazn@unal.edu.co
4 Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Carrera 22 Calle 67, Ibagué 730002,
Colombia; marcial.fuentes@unibague.edu.co
* Correspondence: oswaldo.lopez@unibague.edu.co (O.L.-S.); jose.garcia@unibague.edu.co (O.G.-B.);
Tel.: +57-8-2760-010 (O.G.-B.)




Received: 13 December 2019; Accepted: 5 February 2020; Published: 8 February 2020


Abstract: Detection of an environmental contaminant requires the use of expensive measurement

equipment, which limits the realization of in situ tests because of their high cost, their limited
portability, or the extended time duration of the tests. This paper presents in detail the development
of a portable low-cost spectrophotometer which, by using a specialized chemosensor, allows detection
of mercuric ions (Hg2+ ), providing effective and accurate results. Design specifications for all the
stages assembling the spectrophotometer and the elements selected to build them are presented along
with the process to synthesize the chemosensor and the tests developed to validate its performance in
comparison with a high-precision commercial laboratory spectrophotometer.

Keywords: water contaminants; low-cost spectrophotometer; Hg2+ ions; chemosensor; coumarin

1. Introduction
A spectrophotometer is a measuring device for quantitative analysis generally used to characterize
chemical substances by determining the amount of light that is partially absorbed by the analyte present
in solution [1]. They can be classified according to the spectral region of work, such as ultraviolet
spectrophotometer (UV), from 190 nm to 380 nm; visible spectrophotometer (Vis), from 380 nm to
750 nm; and near infrared spectrophotometer (NIR), from 800 nm to 2500 nm [2–4]. According to
their use, they are classified in stationary devices for analysis in laboratories and portable devices for
determination of substances in fieldwork [1].
There are spectrophotometers that work in non-visible ranges of the electromagnetic spectrum,
such as those presented in References [5,6] (UV and NIR, respectively), which use high-cost elements
like diode arrays and charged couple devices (CCD) as sensors in the detecting stage. For the visible
range, there are several devices that include a Light Emitting Diode (LED) as radiation source [7],
mainly because they have low costs and allow easy implementation. It is worth mentioning the
developments presented in References [8,9] which made analyses for three fixed wavelengths and that
reported in References [10–12] for seven fixed wavelengths which can be selected by the user with the
help of mechanical components. Also, a combination of a white LED and interference filters can be

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used for obtaining different wavelengths as in Reference [13]. Additionally, some devices are used
for chemistry education and allow the user to make a spectral sweep between 400 nm and 700 nm
including a rotating diffraction grating [14]. In terms of portable equipment, those implemented
in References [15–21] can also make spectral sweeps by means of the integration of small motors,
light-scattering elements, low-cost light sources, and simple light detectors. Furthermore, there are
commercial and robust devices for use in laboratory such as the Cary 60 UV-Vis (Agilent Technologies,
CA, USA) [22], which allow the user to make many types of tests, obtaining high-quality analyses,
selectivity, and accuracy at a cost of around 30,000 USD. Portable equipment, such as the DR 1900
(HACH, CO, USA) [23], provides versatility and other advantages in fieldwork at costs between 2000
USD and 8000 USD.
Over the last few decades, the impact of heavy metal pollution has been increasing [24].
Mercury is the heavy metal with the greatest impact on the pollution of ecosystems worldwide [25],
contributing to bioaccumulation in different organisms and being present in the food chain [26].
Its oxidation state Hg2+ has a high affinity for sulfhydryl groups present in biomolecules such as
proteins and endogenous thiols [27,28], which makes it possible to associate the accumulation of
this metal to multiple physiological disorders, the best known of which are damages to the kidney,
central nervous, immune, and endocrine systems [29–34]. There are a variety of analytical methods for
determining mercury; however, the vast majority are complex and require a great deal of resources for
implementation, maintenance, and sample analysis. Each of these methods has its own advantages and
limitations. Among the best-known methods for determining mercury are hyper Rayleigh scattering
(HRS), atomic absorption/emission spectrometry, inductively coupled plasma mass spectrometry
(ICP-MS), colorimetric detection, cold vapor atomic fluorescence spectrometry, high-performance
liquid chromatography, ion chromatography, and electrochemical sensing [35–44]. During the last
decades, there has been increasing interest in developing chemical sensors that can interact with the
analyte through desulphurization, chelation, mercury-mediated hydrolysis reactions, and inclusion
in macromolecules assisted by chemosensors [24,27,35,45]. However, this tool complemented with
low-cost UV-Vis spectrophotometry provides potential alternatives for countries which cannot have
access to expensive instrumentation and sophisticated operating processes.
This paper presents the development of a portable low-cost spectrophotometer, which by using
a chemosensor coumarin-derivate allows detection of mercuric ions (Hg2+ ) in an aqueous medium.
The proposed device has important characteristics such as accuracy, low cost, and portability. The rest
of the paper is organized as follows: Section 2 presents the components required to build the
spectrophotometer, the required embedded digital control algorithms, the methods related with the
synthesis of the chemosensors, and the process necessary to perform each test. After that, Section 3
presents results obtained using the proposed device in comparison with a commercial device and
provides a discussion about how the effectiveness and accuracy of the built device enables its potential
real application. Finally, conclusions are presented in Section 4.

2. Materials and Methods

2.1. Reagents
All analytes were purchased from Sigma-Aldrich and Merck and were used as received.
The metal salts used were HgCl2, CdCl2 , PbCl2 , CaCl2 , MgCl2 , ZnCl2 , FeNH4 (SO4 )2 ·12H2 O,
Fe-(NH4 )2 (SO4)2 ·6H2 O, CuCl2 , MnCl2 , and CoCl2 . All solutions employed in this work were
prepared in EtOH-H2 O (20/80). 1 H and 13 C NMR spectra were recorded on an Avance II 400 MHz
multidimensional spectrometer (Bruker Corporation, Billerica, MA, USA), using the solvent or the
Tetramethylsilane (TMS) signal as an internal standard. High-resolution mass spectra were recorded on
a Pegasus GC×GC (Leco Corporation, St. Joseph, MI, USA), GC-MS system with time of flight analyser,
with 40,000 resolution. Absorption spectra were obtained by means of a Cary 60 spectrophotometer
(Agilent Technologies, Santa Clara, CA, USA).
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2.2. Chemosensor
2.2. Chemosensor Development
Development
The compound
The compoundwas was prepared
preparedusing a conventional
using three-step
a conventional synthesis
three-step from commercial
synthesis precursors
from commercial
until 7-(diethylamine)-2H-chromen-2-one (2) was obtained, followed by
precursors until 7-(diethylamine)-2H-chromen-2-one (2) was obtained, followed by thionation with thionation with Lawesson´s
reagent (Merck-Darmstadt,
Lawesson´s Germany.)Germany.)
reagent (Merck-Darmstadt, as reported in the literature
as reported to obtain
in the literature the the
to obtain compound
compound 7-
(diethylamino)-2H-chromene-2-thione
7-(diethylamino)-2H-chromene-2-thione (3) [46]. (3) [46].
Synthesis of
Synthesis of 7-(diethylamine)-2H-chromen-2-one
7-(diethylamine)-2H-chromen-2-one (2)(2)
(9.8692
(9.8692 g, g,
87.84 %).%).
87.84
1H 1NMR
H NMR (DMSO-d 6) δ 7.50
(DMSO-d 6) δ
(d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 6.53 (dd, J = 12.0 Hz, J = 2.0 Hz, 1H), 6.44 (d,
7.50 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 6.53 (dd, J = 12.0 Hz, J = 2.0 Hz, 1H), 6.44 (d, J = 2.0 J = 2.0 Hz, 1H); 5.99
(d, J1H);
Hz, = 8.05.99
Hz, (d,
1H),J =
3.42
8.0(q,
Hz,J =1H),
8.0 Hz,
3.424H),
(q, J1.17
= 8.0(t,Hz,
J = 8.0 Hz,
4H), 6H).
1.17 (t, JC=NMR
13 (DMSO-d
8.0 Hz, 6): 162.7, 157.1, 151.1,
6H). 13 C NMR (DMSO-d6 ):
144.1, 157.1,
162.7, 129.2, 109.5,
151.1, 109.07, 108.5, 109.5,
144.1, 129.2, 97.9, 45.2, 12.8.108.5, 97.9, 45.2, 12.8.
109.07,
Synthesis of
Synthesis of 7-(diethylamino)-2H-chromene-2-thione
7-(diethylamino)-2H-chromene-2-thione (3) (1.455 g,
(3) (1.455 g, 62.47%).
62.47%). 11H NMR (DMSO-d
H NMR (DMSO-d6)) δδ
6
7.31–7.29 (m, 2H), 6.96 (d, J = 12, 1H), 6.66–6.64 (m, 2H). 13C NMR (DMSO-d6): 197.8, 159.6, 151.3 136.1,
7.31–7.29 (m, 2H), 6.96 (d, J = 12, 1H), 6.66–6.64 (m, 2H). C NMR (DMSO-d6 ): 197.8, 159.6, 151.3 136.1,
13
128.9, 123.3,
128.9, 123.3, 110.8,
110.8, 110.0,
110.0, 97.3,
97.3,45.0,
45.0,12.4.
12.4.High-resolution
High-resolution electrospray
electrospray ionisation
ionisation mass
mass spectrometry
spectrometry
(HRESIMS) was consistent with the molecular formula of C 9H14OS (required: m/z obsd 233.0868; calcd for
(HRESIMS) was consistent with the molecular formula of C9 H14 OS (required: m/z obsd 233.0868; calcd
233.0869).
for 233.0869).

2.3. Determining the Quantum Yield

2.3. Yield of
of Emission
Emission
Fluorescence quantum
Fluorescence quantum yield
yield of
of 33 was
was measured
measured using
using aa solution
solution of
of Rhodamine
Rhodamine 101 101 in
in ethanol
ethanol as
as
standard (Φs =
standard (Φs = 1). All
All values
values were corrected considering the solvent solvent refraction
refraction index.
index. Quantum
Quantum yield
yield
was calculated
was calculated using
using Equation
Equation (1), where the subscripts x and s denote sample and standard,
respectively; φ
respectively; Ф is
is the
the quantum
quantum yield;
yield; ηη is
is the
the refractive
refractive index;
index; and
and Grad
Grad is
is the
the slope
slope from
from the
the plot
plot of
of
integrated fluorescence intensity vs. absorbance
integrated fluorescence intensity vs. absorbance [35]. [35].

Φ = Φ
x ηx
(1)
! 2!
Grad
Φx = Φs (1)
Grads η2s
2.4. Detection Limit
2.4. Detection Limit
The detection of limit (LOD) was calculated based on absorbance and fluorescence titrations. To
The detection of limit (LOD) was calculated based on absorbance and fluorescence titrations.
determine the signal to noise ratio, the absorbance (at 388 nm) of 3 with Hg2+ was measured three
To determine the signal to noise ratio, the absorbance (at 388 nm) of 3 with Hg2+ was measured
times and the standard deviation of calibration curve was determined. The detection limit was
three times and the standard deviation of calibration curve was determined. The detection limit was
calculated with the equation LOD = 3 σb/m, where σb is the standard deviation of calibration curve
calculated with the equation LOD = 3 σb /m, where σb is the standard deviation of calibration curve
and m is the slope of the plot of absorbance or fluorescence intensity versus analyte concentration
and m is the slope of the plot of absorbance or fluorescence intensity versus analyte concentration [35].
[35].
2.5. Electronic Components
2.5. Electronic Components
The proposed low-cost spectrophotometer works in the visible part of the electromagnetic spectrum,
from The proposed
400 nm to 700 nmlow-cost spectrophotometer
(see Figure 1). It is composedworks in the visible
by a low-power part of athe
light source, electromagnetic
monochromator for
spectrum, from 400 nm to 700 nm (see Figure 1). It is composed by a low-power
selecting specific wavelengths, and two light-isolated sample chambers. Each sampling light source,
chamber hasa
monochromator
an for selecting
independent detector whichspecific
allows thewavelengths, and twotolight-isolated
spectrophotometer sample chambers.
make measurements Each
of the reference
sampling chamber has an independent
and the studied substance in a single experiment.detector which allows the spectrophotometer to make
measurements of the reference and the studied substance in a single experiment.

Figure
Figure 1. System architecture
1. System architecture of
of the
the developed
developed spectrophotometer.
spectrophotometer.

2.5.1. Light Source

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Sensors 2020, 20, 906 4 of 16

2.5.1. Light Source

Light Emission Diodes (LEDs) are widely used as light sources in analytical instrumentation
applications [47]. For the proposed device, it is important to use a light source that emits radiation in
the visible region of the electromagnetic spectrum. A 1-W low-cost LED with cold white color with
a 5◦ collimator lens was selected, allowing the radiation pattern to be concentrated and reflected in
a small portion of the diffraction grating, reducing any light power while the test is made. The relative
intensity of a white LED shows that it has a good response in the visible region. Also, by using the
additional hardware presented in Figure 1, the LEDs provide a practical way to have an economic
alternative for the radiation source with low energy consumption (Supplementary Materials Figure S1).

2.5.2. Monochromator
The main task of the monochromator is to select and transmit one specific wavelength of the
visible spectrum to the sample. This goal is achieved in the proposed device using a National Electrical
Manufacturers Association (NEMA) 17 hybrid stepper motor in micro-stepping configuration together
with a low-cost diffraction grating and a small slit that is used for selecting wavelengths. The angle
displacement of the diffraction grating is performed in steps of 1.8◦ , resulting in 200 steps for a
full 360-degree turn. The Allegro a4988 controller is used in micro-stepping configuration to obtain
a good resolution for sweeping all the wavelengths of the visible spectrum enlarging the number of
steps. As the driver makes 1/16 of a step, the resolution results in 0.1125◦ per step. For details of the
configuration of the driver, see Supplementary Materials Figure S2.
The diffraction grating splits and diffracts the radiation emitted by the white LED into individual
wavelengths [48]. Unlike other dispersive elements such as mirrors and prisms, diffraction gratings
are elements with good performance and lower costs. These are composed of a large number of
small parallel slits that have equal spacing between them along the grating and which are capable
of diffracting the incident light that falls on them. Depending on the overtone and the incident
angle of the light beam of the source projected over the grating, an individual color can be observed.
In the developed system, a diffraction grating obtained from a Digital Versatile Disc (DVD) without
information is used. This is a low-cost solution, which contains grooves with a total of 1350 lines/mm
and a separation between them of 780 nm, properties that provide good dispersion and resolution (see
Table 1).

Table 1. Grating separation for different diffraction gratings.

Grating Separation
Grating Lines (inch) Lines (mm)
(nm)
CD 15,875 625 1600
DVD 34,300 1350 740
Lab grade diffraction grating 34,300 1000 1000

The diffraction grating is located in such a way that the range of displacement angles reflects in the
samples the components corresponding to the third overtone produced by the reflection effect (Figure 2)
with the aim of providing a greater spectral resolution in the visible range. Using the micro-stepping
configuration available in the a4988 driver, in which one step is reduced to an advance of 0.1125◦ ,
the 300-nm spectrum is converted into 198 steps, resulting in a selectivity for the monochromator
of 1.52 nm per step. To find the wavelength of a specific color, the following expression is used
(Fraunhofer equation):
nλ = d sin θ, (2)

where λ is the wavelength of a specific color; n = 1 (grating order); d = 740 (distance between slits in
a DVD); and θ = diffraction angle.
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Selected overtone
Figure 2. Selected overtone produced by the diffraction grating.

Table 2 shows the values of the incident angle in the diffraction grating for different colors.

Table 2.
Table Incident angle
2. Incident angle in
in the
the diffraction
diffraction grating
grating for
for obtaining
obtaining different
different colors.
colors.
Color Color Wavelength ◦
Wavelength (nm)(nm) Deflection
Deflection angle (°) Angle ( )
red red 650 650 61 61
orangeorange 600 600 54 54
yellow 575 51
yellow 575 51
green 550 48
blue-greengreen 550 500 48 42.5
blueblue-green 500 450 42.5 37.5
violet blue 450 400 37.5 32.7
violet 400 32.7
2.5.3. Detector
2.5.3. Detector
The main function of a detector is to convert a light signal into an electric signal [49]. The TEMT6000
The light
ambient mainsensor
function of a detector
(Vishay is to convert
Intertechnologies, PA, aUSA)
light issignal
used into
for thean detection
electric signal
stage.[49].
ThisThe
is
TEMT6000 ambient light sensor (Vishay Intertechnologies, PA, USA) is used
a surface mount device that is composed of an Negative Positive Negative (NPN) phototransistor for the detection stage.
This is aonsurface
mounted a printedmount
circuitdevice that is
board (PCB) of 1composed
cm × 1 cm.ofItsan Negative
relative spectral Positive
response Negative
is similar(NPN)
to the
phototransistor
human eye, havingmounted
good on a printedincircuit
sensitivity boardrange
the visible (PCB)of of the
1 cm × 1 cm. Its relative
electromagnetic spectral(Figure
spectrum response 3).
is similar to the human eye, having good sensitivity in the visible range of the
The analog voltage output is proportional to the received amount of light. This voltage is conditioned electromagnetic
spectrum
before (Figure
being 3).the
sent to Themicrocontroller,
analog voltage using
outputa non-inverting
is proportionalamplifier
to the received
of gainamount of light. This
5. The detectors are
voltage is
located in conditioned
such a way thatbefore being
they cansent to the
receive themicrocontroller, using a non-inverting
resulting monochromatic amplifier
radiation after having of passed
gain 5.
The detectors
through are located
the sample andinreference
such a way that they stored
substances can receive
in thethequartz
resulting monochromatic
cuvettes. radiation
Each position after
covered
having
by passed through the
the monochromator sample andto
corresponds reference
a level substances
of voltage stored
in the in the quartz
detector, cuvettes. Each
proportional position
to the light
covered by the monochromator corresponds to a level
transmitted through the reference and sample studied substances.of voltage in the detector, proportional to the light
transmitted through the reference and sample studied substances.

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Figure 3. Details of the detector stage.

Figure 3. Details of the detector stage.
Based on Beer Lambert’s law, the concentration value of a chemical substance is directly
proportional
Based onto Beer
the amount of light
Lambert’s law,it the
absorbs when exposed
concentration valuetoofmonochromatic radiation.isOnce
a chemical substance the
directly
voltage measurements
proportional corresponding
to the amount to the light
of light it absorbs whencaptured
exposedintothe detector for the
monochromatic referenceOnce
radiation. and the
substance being studied are obtained, the following expression is used to find the absorbance
voltage measurements corresponding to the light captured in the detector for the reference and value:
the
substance being studied are obtained, the following expression is used to find the absorbance value:
P
A = log10P = − log10 (T) (3)
A = log Po
= −log (T) (3)
Po
where A is absorbance; P is magnitude of transmitted light through the sample; Po is magnitude of
where A is absorbance; P is magnitude of transmitted light through the sample; Po is magnitude of
transmitted light through the reference; and P/Po = T, which is transmittance.
transmitted light through the reference; and P/Po = T, which is transmittance.
Using voltage levels, we have
Using voltage levels, we have !
P Vsample
A = log10P Vsample
= − log10 (4)
A = log P0= −log ( V solvent ) (4)
P0 V solvent
Table 3 presents the
the most
most absorbed
absorbed color
color in
in every
every produced
produced wavelength.
wavelength.

Table 3. Light absorbance for wavelengths in visible spectra.

Wavelength
Wavelength Absorbed
Absorbedcolor
Color Solution color Solution Color
380–435 380–435 Violet
Violet Green-yellow Green-yellow
435–480 435–480 Blue
Blue Yellow Yellow
480–490 480–490 Blue-green
Blue-green Orange Orange
490–500 490–500 Green-blue
Green-blue Red Red
500–560 Green Violet
500–560 Green Violet
560–580 Green-yellow Violet
580–595 560–580 Green-yellow
Yellow Violet Blue
595–650 580–595 Yellow
Orange Blue Blue-green
650–780 595–650 Red
Orange Blue-green Green-blue
650–780 Red Green-blue
2.5.4. Microcontroller and Control Algorithms
2.5.4. Microcontroller and Control Algorithms
An Atmega 138p microcontroller (Atmel Corp., San Jose, CA, USA) with an Arduino interface
was An Atmega
used 138p microcontroller
to implement the control (Atmel Corp.,required
algorithms CA, USA) bywith
the an Arduino interface operation.
spectrophotometer was used
to implement
First, the control
an initialization routinealgorithms
is performed,required
which byconsists
the spectrophotometer
in finding an initial operation.
positionFirst, an
for the
initialization routine is performed, which consists in finding an initial
monochromator and using a physical reference with a limit switch that guarantees that all tests start at position for the
monochromator
the same wavelength.and using
Then,a using
physical reference withthe
micro-stepping, a limit
stepperswitch
motorthatsweeps
guaranteesall thethat all tests start
wavelengths of
at
thethe samespectrum.
visible wavelength. Then,
In each using
step, themicro-stepping, the stepper
voltage value produced motor
by the sweeps
detector all the wavelengths
is obtained, measuring
of
thethe visiblemonochromatic
resulting spectrum. In each lightstep,
that the
has voltage value produced
passed through the sample. by This
the detector
voltage isisread
obtained,
by the
measuring the resulting
analog-to-digital convertermonochromatic light where
of the microcontroller, that hasit ispassed
filteredthrough the sample.
using a simple averageThis voltage
digital filter.
is readthat,
After by the
theanalog-to-digital
absorbance valueconverter
is computed of the
andmicrocontroller,
stored in a table. where it is filtered
The flowchart of using a simple
the developed
average
algorithmdigital filter. in
is presented After that,
Figure 4. the absorbance value is computed and stored in a table. The
flowchart of the developed algorithm is presented in Figure 4.
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Figure 4. Flowchart of the microcontroller algorithm.

Figure 4. Flowchart of the microcontroller algorithm.
2.5.5. Detailed Hardware Setup
2.5.5. Detailed Hardware Setup
The components are assembled in a black box made with polylactic acid (PLA) polymer by means
of 3D Theprinting with a size
components are of 15 cm × in
assembled 22 acm × 8 box
black cm and
madea wall
withand top cover
polylactic acidthickness of 4 mm,
(PLA) polymer by
ideal
means to reduce the incidence
of 3D printing with a of external
size of 15 cmlight
× 22interferences.
cm × 8 cm andInaorder to obtain
wall and goodthickness
top cover light dispersion,
of 4 mm,
the diffraction
ideal to reducegrating is located
the incidence 18 cm from
of external lightthe sample chambers.
interferences. In orderIntoaddition,
obtain goodit has thedispersion,
light ability to
rotate its position to make incident18angles between ◦ ◦
25 andchambers.
70 , which
the diffraction grating is located cm from the sample Inguarantees
addition, itthehasgeneration
the abilityofto
monochromatic
rotate its positionlightto between 400 nmangles
make incident and 700 nm. The
between 25°detectors are in aguarantees
and 70°, which room withthe light insulationof
generation
monochromatic
that light between
reduces the interference 400experiments
in the nm and 700and nm.guarantees
The detectors
zero are in a when
voltage room with light insulation
the radiation source
isthat
off. reduces
Figure 5 the
shows interference
the physicalin the experiments
distribution of theand guarantees zero voltage when the radiation
components.
source is off. Figure 5 shows the physical distribution of the components.

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(a)

(b)

Figure 5. 5.(a)(a)Geometric
Figure Geometricdistribution
distributionofofthe
thespectrophotometer
spectrophotometerhardware;
hardware;(b)(b)dimensions
dimensionsof of
thethe
developed
developedsystem.
system.
2.5.6. Tests Applied to the Developed System
2.5.6. Tests Applied to the Developed System
To test the operation of the developed equipment, an initial test is performed to measure the
To test the operation of the developed equipment, an initial test is performed to measure the
concentration of the sensor with coumarin, as is done in Reference [50]. A solution with ethanol is used
concentration of the sensor with coumarin, as is done in Reference [50]. A solution with ethanol is
as reference, and 4 solutions with coumarin in 4 known concentrations are tested: 5 µM, 10 µM, 15 µM,
used as reference, and 4 solutions with coumarin in 4 known concentrations are tested: 5 μM, 10 μM,
and 20 µM. The second test consists in measuring mercury concentration. Different solutions are
15 μM, and 20 μM. The second test consists in measuring mercury concentration. Different solutions
prepared with known concentrations of 1 µM, 2 µM, 3 µM, and 4 µM, which are added to the coumarin
are prepared with known concentrations of 1 μM, 2 μM, 3 μM, and 4 μM, which are added to the
with the sensor. Figure 6 shows the color change effect in coumarin when detecting mercury. The tests
coumarin with the sensor. Figure 6 shows the color change effect in coumarin when detecting
run in parallel, making comparisons between the results obtained with the developed prototype and
mercury. The tests run in parallel, making comparisons between the results obtained with the
a commercial Cary 60 spectrometer (Agilent Technologies, Santa Clara, CA, USA).
developed prototype and a commercial Cary 60 spectrometer (Agilent Technologies, CA, USA).

3. Results and Discussion

When building the low-cost UV-Vis spectrophotometer, consideration was given to the
development of a chemosensor to evaluate the optical capabilities of the proposed equipment, which
led to a search in the literature to find alternatives for its synthesis. According to that, the developed

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(diethylamino)-2-hydroxybenzaldehyde (1) was condensed with diethyl malonate in a Knoevenagel
condensation and cyclized and decarboxylated in one step to afford 7-(diethylamino)-2H-chromen-2-one
(2). Then, the compound was modified with Lawesson’s reagent (thionation agent) to obtain the
compound
Sensors 7-(diethylamino)-2-chromene-2-thione
2020, 20, x FOR PEER REVIEW (3) (Figure 6) [46]. The final product was characterized 9 of 16
by using
Sensors H 906
2020,120, and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and High Resolution 9Mass of 16
Spectrometry (HRMS) (Supplementary Materials Figures S3–S7).
chemosensor must have the characteristic to react and determine the metal in aqueous medium by
colorimetric and spectrophotometric methods.
Compound 3 was prepared in three synthetic steps (Supplementary Materials Scheme S1). 4-
(diethylamino)-2-hydroxybenzaldehyde (1) was condensed with diethyl malonate in a Knoevenagel
condensation and cyclized and decarboxylated in one step to afford 7-(diethylamino)-2H-chromen-2-one
(2). Then, the compound was modified with Lawesson’s reagent (thionation agent) to obtain the
compound 7-(diethylamino)-2-chromene-2-thione (3) (Figure 6) [46]. The final product was characterized
by using 1H and Figure 13C Nuclear
Figure 6.
6. ChemicalMagnetic
Chemical Resonance
structure
structure of (NMR) spectroscopy and High Resolution Mass
of 7-(diethylamino)-2H-chromene-2-thione.
7-(diethylamino)-2H-chromene-2-thione.
Spectrometry (HRMS) (Supplementary Materials Figures S3–S7).
3. Results and Discussion
The main photophysical constants of compound 3 were determined, showing an excitation band
(λex) When
at 483 nm (λex) and
building the an emission
low-cost band (λ
UV-Vis ex) at 510 nm (Supplementary
spectrophotometer, consideration Materials
was given FiguretoS8); thea
molar extinction
development of a coefficient,
chemosensor which is 15,754
to evaluate theMoptical
-1 cm capabilities
-1 (Supplementary of theMaterials
proposed Figure equipment, S9); awhich quantum led
yield
to of 0.013;
a search andliterature
in the a Stokes shift of 27
to find nm.
alternatives for its synthesis. According to that, the developed
Compound
chemosensor must 3 was
havetested with different
the characteristic tometals
react and (Hgdetermine
2+, Cd2+, Pbthe 2+, Ca 2+, Mg
metal , Zn2+, Fe2+
in2+aqueous , Fe3+, Cuby
medium 2+,

Mn , and Coand
colorimetric
2+ 2+ ). When testing metals,methods.
spectrophotometric it is observed that only mercuric ions react with chemosensor
3 very quickly and
Compound Figure 6. Chemical structureaof
3 selectively,
was prepared showing
in three colorimetric
synthetic steps (Figure 7a), fluorimetric
(Supplementary
7-(diethylamino)-2H-chromene-2-thione. Materials (Figure Scheme 7b), andS1).
spectrophotometric UV-Vis (Figure 8). In reviewing
4-(diethylamino)-2-hydroxybenzaldehyde (1) was condensed the phenomenon with diethyl that malonate
occurred in in the latter result
a Knoevenagel
beingThe notorious,
condensation mainand by addingand
cyclized
photophysical mercuric ionsofincompound
decarboxylated
constants theinsolution
one step oftocompound
3 were afford 3, there is a decrease
7-(diethylamino)-2H-chromen-2-one
determined, showing an excitation in the bandband
of
(2).483 nm
Then, and
the a bathochromic
compound was displacement
modified to
with 513 nm;
Lawesson’sthen,
(λex) at 483 nm (λex) and an emission band (λex) at 510 nm (Supplementary Materials Figure S8); a over time,
reagent it is observed
(thionation that
agent) the
to 513-nm
obtain
bandcompound
the
molar decreases
extinctionand that therewhichis a hypochromatic
7-(diethylamino)-2-chromene-2-thione
coefficient, is 15,754 M-1 cm shift with
(3) an increase
(Figure
-1 (Supplementary 6) of one The
[46].
Materials band in 388
final
Figure nm
product
S9); UV-Vis
a quantum was
[46] (Supplementary
characterized by using 1 H and 13Figure
Materials C Nuclear S10) and
Magnetic an isosbestic
Resonance point
(NMR) at 432.
spectroscopy Using and different
High
yield of 0.013; and a Stokes shift of 27 nm.
concentrations
Resolution
Compound Mass of 3Spectrometry
mercuric
was tested ionswith
(0.25different
(HRMS) to 2.5 μM),
(Supplementary
metalsthe detection , Cdlimit
(Hg2+Materials 2+, Pbwas
Figures
2+, Cacalculated
2+, S3–S7).
Mg2+, Zn as2+1.1, Fe×2+10, Fe-9 M
3+, and
Cu2+a,
limit
Mn of
2+,The
andquantification
main
Co2+). When was
photophysicaltesting calculated
metals, itas
constants ofis3.7 × 10 M.that
compound
observed -9 3In
wereaddition,
only determined,
mercuricwe emphasize
showing
ions react withthat,
an when testing
excitation
chemosensor band
with
(λ
3 ex ) the
at addition
483 nm
very quickly and (λ of
) mercuric
and an and
emission other
band ions
(λ simultaneously,
) at 510 nm no
(Supplementary interference
ex selectively, showing aexcolorimetric (Figure 7a), fluorimetric (Figure 7b), and Materialswas observed.
Figure S8); a molar
To confirm
extinction andUV-Vis
coefficient, elucidate
which(Figure what8).was
is 15,754 MIn−1 happening
cm−1 (Supplementary in the reaction, we
Materials conducted
Figure S9);an experiment for
spectrophotometric reviewing the phenomenon that occurred inathequantum yield
latter result
the0.013;
of
being13 Cnotorious,
NMRand (Supplementary
a Stokes
by adding Materials
shift mercuric
of 27 nm.ionsFigurein the S11).
solution When compound3,3 there
of compound was performed
is a decrease in the
in the absence
band
2+was 2+ , Pb2+[35].
and
of 483 presence
nm andofamercuric
Compound 3 was tested
bathochromic ions, with
adisplacement
desulphurization
different to 513reaction
metals (Hgthen,
nm; , Cd evident
over time,, Cait is2+
By, Mg
observed 2+ Zn
adding , Hg
2+2+,, Fe
that the2+
the , Fe=3+S,
OC
513-nm
2+
carbon 2+
atom, andC2 ofCo 2+
the ).there
thiocoumarin (δ metals,
= 194.7)shift itwas clearly displaced tomercuric
166.0
Cu
band ,decreases
Mn and that When is a testing
hypochromatic is with
observed that only
an increase of one bandppm, 388anm
in ions typical
react C2
with
UV-Vis
carbonyl
chemosensor (OC 3 = O)
very signal
quickly of a
andcoumarin
selectively,(Supplementary
showing a Materials
colorimetric
[46] (Supplementary Materials Figure S10) and an isosbestic point at 432. Using different Figure
(Figure S10).
7a), fluorimetric (Figure 7b),
and spectrophotometric
concentrations of mercuric UV-Vis (Figure
ions (0.25 to 2.5 8).μM),
In reviewing
the detection the limit
phenomenon
was calculated that occurred
as 1.1 × 10 in-9theM andlattera
resultof
limit being notorious, was
quantification by adding mercuric
calculated as 3.7ions × 10in-9 M. the Insolution
addition, of compound
we emphasize 3, therethat, is when
a decrease testing in
the band
with of 483 nmofand
the addition a bathochromic
mercuric and otherdisplacement
ions simultaneously, to 513 nm; nothen, over time,
interference wasit isobserved.
observed that the
513-nm To band
confirm decreases and thatwhat
and elucidate therewasis ahappening
hypochromatic in theshift with an
reaction, weincrease
conducted of one an band
experimentin 388 nm for
UV-Vis
the 13C NMR [46] (Supplementary
(Supplementary Materials Materials Figure
FigureS11). S10)When and an isosbestic3 point
compound at 432. Using
was performed in thedifferent
absence
−9 M and
concentrations
and presence ofofmercuric mercuric ionsa (0.25
ions, to 2.5 µM), the
desulphurization detection
reaction waslimit
evident was[35].
calculated
By adding as 1.1 Hg×2+,10 the OC = S
a limit of quantification × 10 was−9
carbon atom C2 of the was calculated(δ
thiocoumarin as =3.7194.7) M.clearly
In addition, we emphasize
displaced to 166.0 ppm, that, when
a typical testingC2
with
carbonylthe addition
(OC = O)ofsignal mercuric and other (Supplementary
of a coumarin ions simultaneously, no interference
Materials Figure S10). was observed.

(a) (b)
Figure 7. (a) Colorimetric change of compound 3 in the presence of mercuric ions; (b) fluorimetric
change of compound 3 in the presence of mercuric ions.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors

(a) (b)
Figure 7. (a) Colorimetric
Colorimetric change
change of
of compound
compound 3 in the presence of mercuric ions; (b) fluorimetric
change of compound 3 in the presence of mercuric
mercuric ions.
ions.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors

Sensors 2020, 20, 906 10 of 16
Sensors 2020, 20, x FOR PEER REVIEW 10 of 16

Sensors 2020, 20, x FOR PEER REVIEW 10 of 16

−6 M) upon addition of ions (1 × 10−4 M) in

× ×1010
Figure8.8.Changes
Figure Changesininthe
theabsorbance
absorbancespectra
spectraofof3 3(5(5 -6 M) upon addition of ions (1 × 10-4 M) in

HH22O:EtOH 80:20.
O:EtOH 80:20.

To confirm and elucidate what was happening in the reaction, we conducted an experiment for
By validating the response of the Cary 60 spectrophotometer (Agilent Technologies, CA, USA)
the 13 C NMR (Supplementary Materials Figure S11). When -6compound 3 was performed in the absence
and the built
Figure low-costinspectrophotometer,
8. Changes the absorbance spectra using
of 3 (5both
× 10 equipment,
M) upon additionthe length
of ionsof(1absorption
× 10-42+M) in was
and presence of mercuric ions, a desulphurization reaction was evident [35]. By adding Hg , the OC
determined
H2O:EtOH reporting
80:20. a maximum of at 483 nm. In Figure 9, it can be clearly seen that the absorption
= S carbon atom C2 of the thiocoumarin (δ = 194.7) was clearly displaced to 166.0 ppm, a typical C2
bands are very similar between both instruments. This gives a clear idea of the approach in the correct
carbonylBy (OC = O) signal
validating of a coumarin
thedevice.
response (Supplementary
of the Cary Materials Figure S10).
60 spectrophotometer
operation of the built Although the response presented by(Agilent Technologies,
the low-cost CA, USA)
spectrophotometer
By
andsome validating
the built the response of the Cary 60 spectrophotometer (Agilent Technologies, Santa Clara,
has noise low-cost
comparedspectrophotometer, using both equipment,
to the Cary 60 spectrophotometer the length ofCA,
(Agilent Technologies, absorption
USA) and was
it
CA, USA)
determined and the built
reporting low-cost
a maximum spectrophotometer, using both equipment, the length of absorption
should be further reduced, this of at 483
will nm. that
imply In Figure 9, it canelectronic
additional be clearly circuitry
seen that the
or absorption
additional
was determined
bands reporting
are veryrequirements a maximum
similar between both of at 483 nm. In
instruments. ThisFigure
gives9, it can be clearlytheseen that theinabsorption
computational have a direct impact on the costaofclear
the idea
deviceof or approach
the duration thethe
of correct
test,
bands are very similar between both instruments. This gives a clear idea of
operation of the built device. Although the response presented by the low-cost spectrophotometer the approach in the correct
respectively.
operation
has someof the built
noise device.
compared Although
to the Cary 60the response presented
spectrophotometer by theTechnologies,
(Agilent low-cost spectrophotometer
CA, USA) and it
has some be
should noise compared
further to thethis
reduced, Carywill60 spectrophotometer
imply that additional (Agilent Technologies,
electronic Santa
circuitry or Clara, CA,
additional
USA) and it should be further
0,60 reduced, this will imply that additional electronic
computational requirements have a direct impact on the cost of the device or the duration of the test, circuitry or additional
computational
respectively. requirements have a direct impact on the cost of the device or the duration of the
2 3 [20 μM]
test, respectively. 1
0,45
0,60
2 3 [15 μM]
AbsorbanceAbsorbance

2 1 3 [20 μM]
0,30 1
0,45
2 3 [10 μM]
1
0,15 2 3 [15 μM]
1
0,30 2 3 [5 μM]
1
2 3 [10 μM]
0,00 1
0,15 400 450 500 550
2 (nm)
Wavelength 3 [5 μM]
1

0,00
Figure 9. Comparison of the absorbance
400
responses
450
of compound
500
3 using (1) the spectrophotometer
550
Cary 60 and (2) the low-cost spectrophotometer in H2O:EtOH 80:20.
Wavelength (nm)
In contrast,
Figure Table 4 shows
9. Comparison the comparison
of the absorbance between
responses the measurements
of compound made
3 using (1) the using both devices.
spectrophotometer
It is Cary
important
Figure 9. to note
Comparison the absorbance
of the measurements
absorbance responses ofwith error
compound
60 and (2) the low-cost spectrophotometer in H2 O:EtOH 80:20. percentages
3 using (1) lower
the than 10%, which
spectrophotometer
are similar
Cary 60in andboth instruments.
(2) the The measurement
low-cost spectrophotometer in H2with
O:EtOHthe80:20.
error percentage higher than 10% is
obtained in the lowest coumarin concentration, which is seen as an opportunity to improve the
In contrast,
sensitivity of the Table 4 shows
detectors thethey
so that comparison between
have better the measurements
performances made conditions.
at these specific using both devices.
It is important to note the absorbance measurements with error percentages lower than 10%, which
are similar
Sensors 2020, 20,in both
x; doi: FORinstruments.
PEER REVIEWThe measurement with the error percentage higher than 10% is
www.mdpi.com/journal/sensors
obtained in the lowest coumarin concentration, which is seen as an opportunity to improve the
sensitivity of the detectors so that they have better performances at these specific conditions.
Sensors 2020, 20, 906 11 of 16

In contrast, Table 4 shows the comparison between the measurements made using both devices.
It is important to note the absorbance measurements with error percentages lower than 10%, which are
similar in both instruments. The measurement with the error percentage higher than 10% is obtained
in the lowest coumarin concentration, which is seen as an opportunity to improve the sensitivity of the
Sensors 2020, 20, x FOR PEER REVIEW 11 of 16
detectors so that they have better performances at these specific conditions.
Table 4. Measurements of coumarin concentration.
Table 4. Measurements of coumarin concentration.
Absorbance Absorbance Cary Measurement Error percentage
Concentration Absorbance Absorbance Cary Measurement Error Percentage
Concentration prototype
Prototype
60 60
difference
Difference
(%)
(%)
5 μM 0.109 0.095 0.014 12.84
5 µM 0.109 0.095 0.014 12.84
1010μM
µM 0.232 0.232 0.226 0.226 0.006
0.006 2.65
2.65
1515μM
µM 0.385 0.385 0.360 0.360 0.025
0.025 6.94
6.94
2020μM
µM 0.532 0.532 0.532 0.532 0.000
0.000 0.0
0.0

With the obtained results, the calibration curve of the instrument for the case of coumarin
With the obtained
concentration results,
is computed. This the calibration
is presented curve of
in Figure 10.the instrument for the case of coumarin
concentration is computed. This is presented in Figure 10.

Figure 10. Calibration curve for coumarin concentration.

Figure 10. Calibration curve for coumarin concentration.
Just like the previous test, both the prototype and the commercial device show the same absorption
band Just
characteristics as compound
like the previous by adding
test, 3;both different concentrations
the prototype of mercuric
and the commercial ions,
device the increasing
show the same
behavior of a new absorption band is evident. This displacement occurs by a desulphurization
absorption band characteristics as compound 3; by adding different concentrations of mercuric ions,
reaction [35] when
the increasing Hg2+ reacts
behavior withabsorption
of a new the sulfur of thiocarbonyl
band is evident.of This
compound 3, additionally
displacement occurs byanda
perceptible changing
desulphurization to the human
reaction [35] when eye Hg
given colorimetric
2+ reacts with thechanges
sulfurofofyellow to pink. of
thiocarbonyl Likewise,
compoundit can
3,
be seen in Figure 11 how the signal forms are very similar between the reported in both
additionally and perceptible changing to the human eye given colorimetric changes of yellow to pink. instruments.
There are some
Likewise, it canvariations
be seen inthat are 11
Figure identified
how theinsignal
the lower
formspart
are of thesimilar
very signal between
given by the
the reported
prototype,in
which can be corrected
both instruments. Therebyare
implementing
some variations additional filters.
that are identified in the lower part of the signal given
by the prototype, which can be corrected by implementing additional filters.

(a) (b)
the increasing behavior of a new absorption band is evident. This displacement occurs by a
desulphurization reaction [35] when Hg2+ reacts with the sulfur of thiocarbonyl of compound 3,
additionally and perceptible changing to the human eye given colorimetric changes of yellow to pink.
Likewise, it can be seen in Figure 11 how the signal forms are very similar between the reported in
both instruments.
Sensors 2020, 20, 906 There are some variations that are identified in the lower part of the signal12given
of 16
by the prototype, which can be corrected by implementing additional filters.

Sensors 2020, 20, x FOR PEER REVIEW 12 of 16

(a) (b)
Figure11.
Figure 11.Absorbance
Absorbancethethecompound
compound3 3and
andresponse
responseininfront
frontmercuric
mercuricions:
ions:(a)
(a)spectrophotometer
spectrophotometer
Cary®®60;
Cary 60;(b)
(b)the
thelow-cost
low-costspectrophotometer
spectrophotometerininHH22O:EtOH
O:EtOH80:20.
80:20.
Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors
Table5 5shows
Table showsthe thecomparison
comparisonbetween
betweenthe theresults
resultsobtained
obtainedfrom
fromboth
bothcompared
compared devices.
devices.
Percentageerrors
Percentage errorsininthe
theabsorbance
absorbancemeasurements
measurementsareareless
lessthan
than5%,
5%,which
whichshows
showsthe
theaccuracy
accuracyininthe
the
detectionofofthe
detection theconcentration
concentrationofofmercury
mercuryions.
ions.

Table5.5.Measurements
Table Measurementsofofmercury
mercuryconcentration.
concentration.

Absorbance
AbsorbanceAbsorbance Cary Cary Measurement
Absorbance Measurement Error percentage
Error Percentage
Concentration
Concentration
Prototype 60 Difference (%)
prototype 60 difference (%)
11 μM
µM 0.0660.066 0.065 0.065 0.001
0.001 1.54
1.54
22 μM
µM 0.1280.128 0.132 0.132 0.004
0.004 3.03
3.03
33 μM
µM
0.1620.162 0.169 0.169 0.007
0.007 4.14
4.14
4 µM 0.195 0.194 0.001 0.52
4 μM 0.195 0.194 0.001 0.52

Withthe
With theobtained
obtainedresults,
results,the
thecalibration
calibrationcurve
curveofofthe
theinstrument
instrumentfor
forthe
thecase
caseofofmercury
mercury
concentrationisiscomputed.
concentration computed.This
Thisisispresented
presentedininFigure
Figure12.
12.

Figure 12. Calibration curve for Hg2+ at different concentrations.

Figure 12. Calibration curve for Hg2+ at different concentrations.
In addition to its good performance, the total cost of the device deserves special attention since it
is lower
In than 86 USD.
addition to itsCost
gooddiscriminated
performance,bythe
components
total cost ofisthe
presented in Table 6.special attention since
device deserves
it is lower than 86 USD. Cost discriminated by components is presented in Table 6.

Table 6. Total cost of the developed spectrophotometer.

Component Cost (USD)

Light source 2
Stepper motor 14
Arduino UNO 21
Sensors 2020, 20, 906 13 of 16

Table 6. Total cost of the developed spectrophotometer.

Component Cost (USD)

Light source 2
Stepper motor 14
Arduino UNO 21
Stepper driver 4
Light detectors 10
Black Box 20
Additional elements 15
Total 86

4. Conclusions
A portable low-cost spectrophotometer was developed and described in detail in this paper.
The use of the proposed device has been focused on but is not limited to detection of mercuric ions
Hg2+ in an aqueous medium. A particular feature to highlight of the proposed device and its test
method is the use of a chemosensor which allowed the determination of mercuric ions in micromolar
concentrations by means of a desulphurization reaction which was characterized by spectrophotometric
methods and through 13 C NMR spectroscopy. When evaluating the developed device, only slight
differences were obtained between the results of the proposed device and the equivalent equipment
used in a traditional laboratory. Future efforts will concentrate on extending the possible application
of the device to analysis of quality in fuels, determination of plasma glucose, color measurement,
and even forensic applications. This work demonstrates how interdisciplinary interaction between
electronics engineering and basic sciences can provide solutions facilitating a wide research and
academic community having access to specialized test like this.

Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/20/3/906/s1,

Figure S1: (a) 1W used white LED. (b) 5◦ collimator lens, Figure S2: a4988 driver in microstepping
configuration, Figure S3: 1 H NMR (DMSO-d6 ) spectrum of 7-(diethylamino)-2H-chromen-2-one (2), Figure S4:
13 C NMR (DMSO-d ) spectrum of 7-(diethylamino)-2H-chromen-2-one (2), Figure S5: 1 H NMR (DMSO-d )
6 6
spectrum of 7-(diethylamino)-2H-chromene-2-thione (3), Figure S6: 13 C NMR (DMSO-d6 ) spectrum of
7-(diethylamino)-2H-chromene-2-thione (3), Figure S7: HRMS of 7-(dietilamino)-2H-cromeno-2-tiona (3), Figure
S8: UV-Vis and emission spectra of 3, Figure S9: (A) UV-Vis spectra of 3 at different concentration; (B) Plot of
absorbance of chemosensor 3 against its concentration from 3 to 30 µM, Figure S10: (A) UV-Vis spectra of 3
at different concentration; (B) Plot of absorbance of chemosensor 3 against its concentration from 3 to 30 µM,
Figure S11: 13 C NMR (DMSO-d6 ) spectrum of chemosensor 3 alone (A) and in the presence of mercuric ions (B),
Scheme S1: Synthetic route to 7-(diethylamino)-2-chromene-2-thione (3).
Author Contributions: Conceptualization, D.G.-M., O.L.-S. and O.G.-B.; Formal analysis, D.G.-M., M.F.-E.,
A.D.-N., A.V., O.L.-S. and O.G.-B. Funding acquisition, O.G.-B. and O.L.-S. Investigation, D.G.-M., M.F.-E., A.D.-N.,
A.V., O.L.-S. and O.G.-B.; Methodology, D.G.-M., O.L.-S. and O.G.-B.; Project administration, O.G.-B.; Validation,
D.G.-M., M.F.-E., A.D.-N., A.V., O.L.-S. and O.G.-B.; Writing—Original Draft, D.G.-M.; Writing—Review and
Editing, D.G.-M., O.L.-S. and O.G.-B. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Departamento Administrativo de Ciencia, Tecnología e Innovación
(COLCIENCIAS) under grant number 130774559056.
Acknowledgments: This work was supported by COLCIENCIAS Grant #130774559056-Colombia.
Conflicts of Interest: The authors declare that there is no conflict of interest.

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